Biomedical subjects

Patrizia Ferretti

Publications and source records attributed to Patrizia Ferretti.

15 recordsLinked to original sources

PubMed2006

Growth of choroid plexus epithelium vesicles in vitro depends on secretory activity.

Although a number of models have been used to study choroid plexus epithelium (CPe) function, analysis in physiological conditions of this polarised epithelium which produces the majority of the cerebrospinal fluid (CSF) and is one of the key barriers between blood and CSF in the brain remains challenging. As CPe cells form polarised CPe vesicles when cultured in Matrigel, we have assessed their behaviour and potential use for pharmacological studies. Like CPe cells in vivo, CPe vesicles express transthyretin, E2f5, Fox-j1 and p73, and contain tight junctions, as indicated by ZO-1 expression and electron microscopy analysis. Time-lapse microscopy shows that CPe cells plated in Matrigel are highly migratory and rapidly form homotypic cell aggregates, which then reorganise to form vesicles whose size increases linearly overtime. Neither aggregate nor vesicle size is affected by AraC treatment, though this inhibitor significantly reduces proliferation in CPe monolayers. Increase in size of vesicles, which have reached a growth plateau is observed following addition of fluorescently-labelled CPe cells, which become incorporated into the vesicle walls. Significantly, treatment with secretion inhibitors blocks vesicle formation and their expansion. These results show that secretion, rather than cell division, controls vesicle growth, consistent with low levels of proliferation and thinning of the CPe observed both in growing vesicles and during CPe development. Therefore, changes in vesicle size can be used to evaluate the effect of putative molecules involved in the regulation of secretion.

Animals↗

PubMed2006

The developing human spinal cord contains distinct populations of neural precursors.

It is becoming apparent that neural stem cells display some differences in their behaviour depending on the region of the CNS they originate from and on whether they are derived from embryonic or adult tissue. Whereas much work has focused on brain neural stem cells, less attention has been paid to spinal cord neural precursors, particularly in the developing human embryo. We briefly review here some of our work which points at some similarities between neural precursors in developing human spinal cords and in animals which can regenerate their spinal cord (e.g. tailed amphibians), and at differences in the properties of human neural precursors with spinal cord development. Altogether these studies suggest the existence of dynamic neural stem cell populations within the developing spinal cord. They also support the notion that thorough characterization of neural stem cells under different culture conditions and analysis of how these may affect their differentiation in vivo after grafting into different injury models is imperative if we are to develop effective cell therapy strategies for spinal cord injury and diseases.

Animals↗

PubMed2005

Eocene bipolar glaciation associated with global carbon cycle changes.

The transition from the extreme global warmth of the early Eocene 'greenhouse' climate approximately 55 million years ago to the present glaciated state is one of the most prominent changes in Earth's climatic evolution. It is widely accepted that large ice sheets first appeared on Antarctica approximately 34 million years ago, coincident with decreasing atmospheric carbon dioxide concentrations and a deepening of the calcite compensation depth in the world's oceans, and that glaciation in the Northern Hemisphere began much later, between 10 and 6 million years ago. Here we present records of sediment and foraminiferal geochemistry covering the greenhouse-icehouse climate transition. We report evidence for synchronous deepening and subsequent oscillations in the calcite compensation depth in the tropical Pacific and South Atlantic oceans from approximately 42 million years ago, with a permanent deepening 34 million years ago. The most prominent variations in the calcite compensation depth coincide with changes in seawater oxygen isotope ratios of up to 1.5 per mil, suggesting a lowering of global sea level through significant storage of ice in both hemispheres by at least 100 to 125 metres. Variations in benthic carbon isotope ratios of up to approximately 1.4 per mil occurred at the same time, indicating large changes in carbon cycling. We suggest that the greenhouse-icehouse transition was closely coupled to the evolution of atmospheric carbon dioxide, and that negative carbon cycle feedbacks may have prevented the permanent establishment of large ice sheets earlier than 34 million years ago.

Calcium Carbonate↗

PubMed2005

FGFR1 down-regulation in differentiating human brain and spinal cord neurospheres.

FGF2 is a key regulator of survival and proliferation of mammalian CNS stem cells. Cells within undifferentiated rodent neurospheres express FGF receptors (FGFRs), but their expression patterns and potential roles in human neurosphere proliferation and differentiation have not been examined. Our aim was to provide an initial overview of the relative profiles of FGFRs before and after differentiation of human neurospheres derived either from embryonic brain or spinal cord. In 'undifferentiated' neurospheres, transcripts from FGFR1 and FGFR2 were consistently detected. FGFR3 could be detected in undifferentiated brain neurospheres and in spinal cord early neurospheres. Following differentiation the most dramatic and consistent change was a decrease in FGFR1 mRNA, suggesting a role for this receptor in maintenance of the undifferentiated state.

Brain↗

PubMed2005

Changes in E2F5 intracellular localization in mouse and human choroid plexus epithelium with development.

The choroid plexus epithelium (CPe) is a specialized epithelium involved primarily in the production of cerebrospoinal fluid (CSF) which is important for maintaining an optimal homeostatic environment for the brain. Although, the physiology of the CPe is fairly well understood, its development has not been thoroughly studied. It has been recently shown that mice lacking functional transcription factors, E2F5, foxJ1 or p73, develop non-obstructive hydrocephalus likely due to CPe dysfunction. We have further studied their expression in the mouse and human developing CPe, focusing particularly on E2F5. We show here that in the mouse E2F5, foxJ1 and p73 transcripts are detectable as soon as the choroid plexuses form. E2F5 protein is also detected as soon as the choroid plexuses are morphologically apparent both in mouse and human, suggesting that its expression is regulated at the transcriptional level. E2F5 protein is down-regulated late in embryogenesis and this coincides with a change in its intracellular localization, from predominantly nuclear to cytoplasmic. The pattern of expression and intracellular localization of E2F5 in vivo does not appear to correlate with that of proliferating CPe cells, as indicated by protein cell nuclear antigen (PCNA) staining, but rather with their maturation, as changes in E2F5 localization from the nucleus to the cytoplasm parallel the morphological change from pseudostratified to cuboidal epithelium.

Animals↗

PubMed2004

Nogo and Nogo-66 receptor in human and chick: implications for development and regeneration.

Antibodies to the myelin protein Nogo increase axonal regrowth after central nervous system injury. We have investigated whether Nogo expression contributes to loss of regenerative potential during development by using chick embryos, which regenerate their spinal cord until embryonic day (E) 13, when myelination begins. We show that Nogo-A and the Nogo receptor (NgR) are developmentally regulated both in chick and human embryos, are first detected at developmental stages when the chick spinal cord regenerates, and are not down-regulated after injury at permissive stages for regeneration. Therefore, expression of Nogo-A and NgR in pre-E13 chick spinal cords is not sufficient to inhibit regeneration. Nogo-A expression in the chick early embryo is primarily observed in axons, whereas NgR is mainly located on neuronal cell bodies, both in spinal cord and eye, and in striated muscle including the heart. With the onset of myelination, there is down-regulation of Nogo-A expression in neurons. Therefore, loss of regenerative potential might be linked to changes in its cellular localization. The possibility that only Nogo expressed in mature oligodendrocytes can exercise inhibitory effects would reconcile the lack of inhibition we observe in developing chick spinal cords before the onset of myelination with evidence from other laboratories on the inhibitory effects of Nogo in mature central nervous system. The distinctive and complementary patterns of Nogo-A and NgR expression and their conservation throughout evolution support the view that Nogo signaling represents a key pathway in nervous system and striated muscle development. Its putative role in target innervation and establishment of neural circuitry is discussed.

Amino Acid Sequence↗

PubMed2004

Distinct neural precursors in the developing human spinal cord.

Both embryonic and adult central nervous system have been shown to contain multipotent neural stem cells, but it is not yet clear whether they consist of a single or distinct populations of neural precursors. Since embryonic human neural precursors, particularly in the spinal cord, have not been extensively characterized, we have studied their behaviour at different days of gestation and in different culture conditions. Depending on dissociation and culture conditions, neurospheres which contain nestin- and vimentin-positive or only vimentin-positive neural precursors can be isolated. Whereas the former can be isolated only at early developmental stages, the latter appear to be present at all the stages examined, between 45 and 89 days of gestation. Furthermore, comparison of the effect of FGF, EGF and the two factors in combination on colony formation shows an additive effect of the two growth factors, indicating the existence of more than one type of neural precursor. Overall our results suggest that the human spinal cord contains distinct and dynamic populations of neural precursors which are developmentally regulated.

Cell Differentiation↗

PubMed2004

Neural stem cell plasticity: recruitment of endogenous populations for regeneration.

Lower vertebrates, such as fish and urodele amphibians can regenerate complex body structures including significant portions of their central nervous system by recruiting progenitor cells to repair the damage. Significant ability to regenerate the nervous system is observed also during development in higher vertebrates, for example in the chick spinal cord, though it is not yet clear whether this involves de novo neurogenesis, in addition to axonal re-growth, also at the latest stages of development permissive for regeneration. The mechanisms underlying recruitment of progenitor cells in response to injury, particularly within the nervous system, are still poorly understood. Although it has been suggested that some neurogenesis can be induced even in regions of the adult mammalian brain, this potential is largely lost with evolution and development. Following tail amputation in urodeles, an ependymal tube, resembling a developing neural tube, forms from ependymal cells that migrate from the cord stump towards the terminal vesicle, and elongates by cell proliferation. The new cord might originate from stem cells, with possibly only a subset of ependymal cells displaying such properties, or via a process of dedifferentiation / transdifferentiation of these cells. Data currently available are more supportive of the latter hypothesis. Whereas dedifferentiation is a well demonstrated phenomenon in a broad range of urodele tissues, transdifferentiation seems to occur less widely and in extreme circumstances, and may contribute significantly to regeneration only in a few cases. In higher vertebrates it is even less clear how common and relevant to repair transdifferentiation is, as much work both in favour and against it has recently been published. However, the existence of multipotent neural progenitors in adult mammalian CNS and of a much higher neural cell plasticity, at least in vitro, than previously believed, encourages the view that if we were to better understand progenitor cell recruitment and plasticity in species where it does occur spontaneously, we might then find the way to make it happen effectively in mammals.

Animals↗

PubMed2003

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord.

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.

Animals↗

PubMed2003

Differential expression of fibroblast growth factor receptors in the developing murine choroid plexus.

The choroid plexuses (CPs) are specialised secretory organs situated within the ventricles of the brain involved in the production of cerebrospinal fluid (CSF) and the maintenance of the blood-CSF barrier. Abnormal function of the CPs can lead to hydrocephalus and raised intracranial pressure, pathologies frequently observed in certain craniofacial syndromes caused by single point mutations in fibroblast growth factor receptors (FGFRs). At present, relatively little is known about the embryonic CPs in terms of gene or protein expression, function as the brain develops or on the potential role of FGFRs within this context. Given the limited information available on the regulation of FGFRs during development of the CPs and periventricular tissues, we have carried out a detailed analysis of the localisation of FGFR1, 2, 3 and 4 proteins in these regions of the murine embryo from the time of formation of the CP in the third ventricle at E12.5 throughout the second half of gestation, and examined the expression of different FGFR isoforms at E12.5 by RT-PCR. We show here that FGFR1 and FGFR4 are expressed in murine CPs at E12.5 but not at E15.5 or E18.5, suggesting a role for the signaling pathways transduced by these receptors at early stages of CP development. In contrast, FGFR2 expression is maintained throughout CP development, indicating that this receptor may play a role in the function of immature and mature CP. Also FGFR3 is detected at each developmental stage studied, but surprisingly its expression appears confined to the nuclei of CP cells, suggesting that FGFR3 in the CP does not respond to extracellular FGFs but may act in intracrine fashion.

Animals↗

PubMed2003

Changes in spinal cord regenerative ability through phylogenesis and development: lessons to be learnt.

Lower vertebrates, such as fish and amphibians, and developing higher vertebrates can regenerate complex body structures, including significant portions of their central nervous system. It is still poorly understood why this potential is lost with evolution and development and becomes very limited in adult mammals. In this review, we will discuss the current knowledge on the cellular and molecular changes after spinal cord injury in adult tailed amphibians, where regeneration does take place, and in developing chick and mammalian embryos at different developmental stages. We will focus on the recruitment of progenitor cells to repair the damage and discuss possible roles of changes in early response to injury, such as cell death by apoptosis, and of myelin-associated proteins, such as Nogo, in the transition between regeneration-competent and regeneration-incompetent stages of development. A better understanding of the mechanisms underlying spontaneous regeneration of the spinal cord in vivo in amphibians and in the chick embryo will help to devise strategies for restoring function to damaged or diseased nervous tissues in mammals.

Animals↗

PubMed2003

Recruitment of postmitotic neurons into the regenerating spinal cord of urodeles.

By using fluorescent tracers, we have investigated the origin of the cells that form the regenerating spinal cord after tail amputation in urodele amphibians. We show that spinal cord cells immediately adjacent to the amputation plane die and are removed by phagocytic cells. Spinal cells just anterior to these dying cells are destined to make the majority of the regenerating cord. The largest contribution is likely to come from the radial ependymal cells, but we also demonstrate that postmitotic neurons in this location can translocate into the regenerating cord. These neurons integrate into the regenerate structure and survive for at least 4 weeks. We find no evidence that these translocated neurons dedifferentiate and divide during this regeneration process. We discuss the possibility that these neurons survive long term in the regenerate cord and become part of the functional neuronal circuitry.

Ambystoma↗

PubMed2002

Blocking endogenous FGF-2 activity prevents cranial osteogenesis.

Normal growth and morphogenesis of the cranial vault reflect a balance between cell proliferation in the sutures and osteogenesis at the margins of the cranial bones. In the clinical condition craniosynostosis, the sutures fuse prematurely as a result of precocious osteogenic differentiation and craniofacial malformation results. Mutations in several fibroblast growth factor receptor (FGFR) genes have now been identified as being responsible for the major craniosynostotic syndromes. We have used a grafting technique to manipulate the levels of endogenous FGF-2 ligand in embryonic chick cranial vaults and thereby perturb morphogenesis. Implantation of beads loaded with FGF-2 did not affect normal cranial development at physiological concentrations, although they elicited a morphogenetic response in the limb. Implantation of beads loaded with a neutralising antibody to FGF-2 generated a concentration-dependent response. When a single bead was implanted, the grafts grew to a massive size as a result of increased cell division in the tissue. With greater inactivation of FGF-2 protein (two to three beads implanted), all further bone differentiation and cell proliferation was blocked. These data further support the emerging idea that the intensity of FGF-mediated signalling determines the developmental fate of the skeletogenic cells in the cranial vault. High and low levels correlate with differentiation and proliferation, respectively. A balance between the two ensures normal cranial vault morphogenesis. This is consistent with the observation that several FGFR mutations causing craniosynostosis result in constitutive activation of the receptor.

Animals↗

PubMed2002

Induction of chondrogenesis in neural crest cells by mutant fibroblast growth factor receptors.

Activating mutations in human fibroblast growth factor receptors (FGFR) result in a range of skeletal disorders, including craniosynostosis. Because the cranial bones are largely neural crest derived, the possibility arises that increased FGF signalling may predispose to premature/excessive skeletogenic differentiation in neural crest cells. To test this hypothesis, we expressed wild-type and mutant FGFRs in quail embryonic neural crest cells. Chondrogenesis was consistently induced when mutant FGFR1-K656E or FGFR2-C278F were electroporated in ovo into stage 8 quail premigratory neural crest, followed by in vitro culture without FGF2. Neural crest cells electroporated with wild-type FGFR1 or FGFR2 cDNAs exhibited no chondrogenic differentiation in culture. Cartilage differentiation was accompanied by expression of Sox9, Col2a1, and osteopontin. This closely resembled the response of nonelectroporated neural crest cells to FGF2 in vitro: 10 ng/ml induces chondrogenesis, Sox9, Col2a1, and osteopontin expression, whereas 1 ng/ml FGF2 enhances cell survival and Sox9 and Col2a1 expression, but never induces chondrogenesis or osteopontin expression. Transfection of neural crest cells with mutant FGFRs in vitro, after their emergence from the neural tube, in contrast, produced chondrogenesis at a very low frequency. Hence, mutant FGFRs can induce cartilage differentiation when electroporated into premigratory neural crest cells but this effect is drastically reduced if transfection is carried out after the onset of neural crest migration.

Alkaline Phosphatase↗

PubMed2002

Heparan sulphate proteoglycans and spinal neurulation in the mouse embryo.

Heparan sulphate proteoglycans have been implicated in the binding and presentation of several growth factors to their receptors, thereby regulating cellular growth and differentiation. To investigate the role of heparan sulphate proteoglycans in mouse spinal neurulation, we administered chlorate, a competitive inhibitor of glycosaminoglycan sulphation, to cultured E8.5 embryos. Treated embryos exhibit accelerated posterior neuropore closure, accompanied by suppression of neuroepithelial bending at the median hinge point and accentuated bending at the paired dorsolateral hinge points of the posterior neuropore. These effects appear specific, as they can be prevented by addition of heparan sulphate to the culture medium, whereas heparitinase-treated heparan sulphate and chondroitin sulphate are ineffective. Both N- and O-sulphate groups appear to be necessary for the action of heparan sulphate. In situ hybridisation analysis demonstrates a normal distribution of sonic hedgehog mRNA in chlorate-treated embryos. By contrast, patched 1 transcripts are abnormally abundant in the notochord, and diminished in the overlying neuroepithelium, suggesting that sonic hedgehog signalling from the notochord may be perturbed by inhibition of heparan sulphation. Together, these results demonstrate a regulatory role for heparan sulphate in mouse spinal neurulation.

Actins↗